No abstract

Lucidone, which comprises a naturally occurring cyclopentenedione, has been investigated for its in vitro and in vivo wound healing properties, and the underlying molecular signaling cascades in the wound healing mechanism have been elucidated. We demonstrated the cell-/dose-specific responses of lucidone (0.5-8μM) on proliferation and migration/invasion of keratinocyte HaCaT and fibroblast Hs68 cells. In keratinocytes, lucidone-induced nuclear translocation of β-catenin was accompanied by increased transcriptional target genes, including c-Myc and cyclin-D1, through GSK3β-dependent pathway. Correspondingly, lucidone promoted the cell-cycle by increasing PCNA/CDK4 and decreasing p21/p27 expressions. Lucidone induced EMT through the downregulation of epithelial (E-cadherin/occludin) and upregulation of mesenchymal (vimentin/Twist/Snail) marker proteins. Activated MMP-9/-2 and uPA/uPAR as well as suppressed TIMP-1/-2 and PAI-1 expressions by lucidone may promote the migration/invasion of keratinocytes. Notably, lucidone activated NF-κB signaling via IKK-mediated-IκB degradation, and its inhibition abolished MMP-9 activation and keratinocyte migration. Inhibition of PIK/AKT signaling impaired the lucidone-induced proliferation/migration with corresponding suppression of β-catenin/c-Myc/cyclin-D1 and NF-κB/MMP-9 expressions. Results indicate that lucidone-induced PIK/AKT signaling anchored the β-catenin/NF-κB-mediated healing mechanism. β-catenin knockdown substantially diminished lucidone-induced keratinocyte migration. Furthermore, lucidone increased endothelial cell proliferation/migration and triggered angiogenesis (MMP-9/uPA/ICAM-1). In macrophages, lucidone-activated NF-κB-mediated inflammation (COX-2/iNOS/NO) and VEGF, which may contribute to the growth of keratinocytes/fibroblasts and endothelial cells. Punched wounds on mice were rapidly healed with the topical application of lucidone (5mM) compared with control ointment-treated mice. Taken together, lucidone accelerates wound healing through the cooperation of keratinocyte/fibroblast/endothelial cell growth and migration and macrophage inflammation via PIK/AKT, Wnt/β-catenin and NF-κB signaling cascade activation.

O. fragrans has slightly less antioxidative activity than green tea. Five phenolic compounds, tyrosyl acetate (1), (+)-phillygenin (2), (8E)-ligustroside (3), rutin (4), and verbascoside (5), were isolated from the CHCl3 sub-extract of O. fragrans. The structures were elucidated by interpreting their spectral data. Evaluation of the antioxidative property of the isolated (+)-phillygenin (2), rutin (4), and verbascoside (5) revealed strong DPPH radical scavenging activity, with IC50 values of 19.1, 10.3, and 6.2 μM, respectively. These isolates also exhibited an H2O2 scavenging ability, with IC50 values of 10.5, 23.4, and 13.4 μM, respectively.

POU1F1, a pituitary-specific POU-homeo domain transcription factor, plays an essential role in the specification of the somatotroph, lactotroph and thyrotroph lineages and in the activation of GH1, PRL and TSHβ transcription. Individuals with mutations in POU1F1 present with combined deficiency of GH, PRL and TSH. Here, we identified a heterozygous missense mutation with evidence of pathogenicity, at the POU1F1 locus, in a large family in which an isolated growth hormone deficiency segregates as an autosomal dominant trait. The corresponding p.Pro76Leu mutation maps to a conserved site within the POU1F1 transactivation domain. Bandshift assays revealed that the mutation alters wild-type POU1F1 binding to cognate sites within the hGH-LCR and hGH1 promoter, but not to sites within the PRL promoter, and it selectively increases binding affinity to sites within the hGH-LCR. Co-immunoprecipitation studies reveal that this substitution enhances interactions of POU1F1 with three of its cofactors, PITX1, LHX3a and ELK1, and that residue 76 plays a critical role in these interactions. The insertion of the mutation at the mouse Pou1f1 locus results in a dramatic loss of protein expression despite normal mRNA concentrations. Mice heterozygous for the p.Pro76Leu mutation were phenotypically normal while homozygotes demonstrated a dwarf phenotype. Overall, this study unveils the involvement of POU1F1 in dominantly inherited isolated GH deficiency and demonstrates a significant impact of the Pro76Leu mutation on DNA-binding activities, alterations in transactivating functions and interactions with cofactors. Our data further highlight difficulties in modeling human genetic disorders in the mouse despite apparent conservation of gene expression pathways and physiologic functions.

The changes in the microenvironment of the Trp-3 on the i-face of pig pancreatic IB phospholipase A2 (PLA2) provide a measure of the tight contact (Ramirez and Jain, Protein Sci. 9, 229-239, 1991) with the substrate interface during the processive interfacial turnover. Spectral changes from the single Trp-substituent at position 1, 2, 6, 10, 19, 20, 31, 53, 56 or 87 on the surface of W3F PLA2 are used to probe the Trp-environment. Based on our current understanding only the residue 87 is away from i-face, therefore all other mutants are well suited to report modest differences along the i-face. All Trp-mutants bind tightly to anionic vesicles. Only those with Trp at 1, 2 or 3 near the rim of the active site on the i-face cause significant perturbation of the catalytic functions. Most other Trp-mutants showed < 3-fold change in the interfacial processive turnover rate and the competitive inhibition by MJ33. Binding of calcium to the enzyme in the aqueous phase had modest effect on the Trp-emission intensity. However, on the binding of the enzyme to the interface the fluorescence change is large, and the rate of oxidation of the Trp-substituent with N-bromosuccinimide depends on the location of the Trp-substituent. These results show that the solvation environment of the Trp-substituents on the i-face is shielded in the enzyme bound to the interface. Additional changes are noticeable if the active site of the bound enzyme is also occupied, however, the catalytically inert zymogen of PLA2 (proPLA2) does not show such changes. Significance of these results in relation to the changes in the solvent accessibility and desolvation of the i-face of PLA2 at the interface is discussed.